title: High-performance affinity purification for identification of 15-deoxy-Δ 12,14 -PGJ₂ interacting factors using magnetic nanobeads.
abstract: Prostaglandin J₂ (PGJ₂) family have been reported to show various kinds of biological activities. Considerable progress has been made toward understanding the mechanism of adipogenesis, however, the mechanisms of other actions of PGJ₂ family remain controversial. The 15-deoxy-Δ(12,14) -PGJ₂ (15d-PGJ₂) is one of the members of PGJ₂ family, and is known as a ligand for peroxisome proliferator-activated receptor γ (PPARγ), which promotes the expression of the crucial genes for adipogenesis. In this study, we found that 15d-PGJ₂ did not stimulate PPARγ-mediated gene expression in HEK293 cells whereas 15d-PGJ₂ transactivated PPARγ-dependent transcription in other cell lines. Moreover, we confirmed that 15d-PGJ₂ suppressed the growth of HEK293 cells. These observations suggest that 15d-PGJ₂ shows another biological activity e.g. growth inhibition in HEK293 cells via unknown receptor for 15d-PGJ₂. The aim of this study is to develop and validate effective purification system for PGJ₂ interacting factors (PGJIFs). We have recently developed high performance magnetic nanobeads. In this study, we have newly developed 15d-PGJ₂-immobilized beads by conjugating 15d-PGJ₂ to the surface of these nanobeads. Firstly, we showed that PPARγ specifically bound to 15d-PGJ₂-immobilized beads. Secondly, we newly identified voltage dependent anionic channel 1 (VDAC1) as new PGJIF from crude extracts of HEK293 cells using this affinity purification system. These data presented here demonstrate that 15d-PGJ₂-immobilized beads are effective tool for purification of PGJIFs directly from crude cell extracts.
authors.affiliation: Department of Aging Intervention, National Center for Geriatrics and Gerontology, Obu, Aichi, Japan.
authors.forename: Naoya
authors.initials: N
authors.lastname: Maekawa
authors.forename: Masaki
authors.initials: M
authors.lastname: Hiramoto
authors.forename: Satoshi
authors.initials: S
authors.lastname: Sakamoto
authors.forename: Motoki
authors.initials: M
authors.lastname: Azuma
authors.forename: Takumi
authors.initials: T
authors.lastname: Ito
authors.forename: Marie
authors.initials: M
authors.lastname: Ikeda
authors.forename: Michitaka
authors.initials: M
authors.lastname: Naitou
authors.forename: Hukum P
authors.initials: HP
authors.lastname: Acharya
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Kobayashi
authors.forename: Makoto
authors.initials: M
authors.lastname: Suematsu
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Handa
authors.forename: Takeshi
authors.initials: T
authors.lastname: Imai
mesh_terms: D048708:Cell Growth Processes / Q000502:physiology; D002846:Chromatography, Affinity / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D047495:PPAR gamma / Q000378:metabolism; D015230:Prostaglandin D2 / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D050995:Voltage-Dependent Anion Channel 1 / Q000378:metabolism
chemical_list: C097240:15-deoxy-delta(12,14)-prostaglandin J2; D008024:Ligands; D058185:Magnetite Nanoparticles; D047495:PPAR gamma; D052247:Nitric Oxide Synthase Type II; D050995:Voltage-Dependent Anion Channel 1; D015230:Prostaglandin D2